The ETS Related Gene (ERG) proto-oncogene was characterized more than twenty-five years ago (Rao et al., 1987a; Rao et al., 1987b; Reddy et al., 1987) and belongs to a large family of ETS transcription factors that are both positive and negative regulators of gene expression (Watson et al., 2010). These transcription factors are downstream effectors of the mitogenic signal transduction pathways involved in cell proliferation, cell differentiation, development, transformation, apoptosis, and immune regulation (Watson et al., 2010; Sreenath et al., 2011; Dobi et al., 2013).
Prostate cancer (CaP) is the most frequently diagnosed non-skin malignancy and second leading cause of cancer related deaths among men in the western countries, with a projected 1.7 million newly diagnosed cases worldwide (International Agency for Research of Cancer, WHO, Press Release No 209, Mar. 21, 2012). An estimated 2.9 million patients in the United States and 11 million world-wide are currently living with prostate cancer (http://globocan.iarc.fr/old/FactSheets/cancers/prostate-new.asp; http://www.cancer.org/cancer/prostatecancer/detailedguide/prostate-cancer-key-statistics). While early detected CaP due to PSA screening is managed effectively by surgery or radiation, a significant subset of CaP patients (20% to 40%) experience disease recurrence after definitive treatment and will require hormone ablation therapy (Eur. Urol. 2007 May; 51(5):1175-84, Epub 2007). Despite an initial response to therapy, metastatic CaP tumors eventually become refractory to hormone ablation therapy. For this subset of patients—i.e., those having metastatic hormone refractory cancer, there is no effective cure.
The ERG gene is the most prevalent and validated genomic alteration in prostate cancer The ERG proto-oncogene is overexpressed in 60-70% of prostate tumors in patients of Caucasian ancestry as a result of recurrent gene fusions involving TMPRSS2 and the ETS family of genes (Petrovics et al., 2005; Tomlins et al., 2005; reviewed in Kumar Sinha et al., 2008; Rubin et al., 2012). Emerging studies on human prostate cancer specimens and various experimental models underscore the causative oncogenic function of ERG in prostate cancer (Klezovitch et al., 2008; Tomlins et al., 2008; Sun et al., 2008; Wang et al., 2008). ETS factors reprogram the androgen receptor cistrome and prime prostate tumorigenesis in response to PTEN loss (Chen et al., 2013; Nguyen et al., 2015) Numerous reports have highlighted both diagnostic and prognostic features of the genomic activation of ERG revealing that about half the prostate tumors harbor the most common gene fusion that takes place between the androgen receptor-regulated TMPRSS2 gene promoter and ERG protein coding sequence (reviewed in Kumar-Sinha et al., 2008; Rubin et al., 2012). Fusion between the TMPRSS2 gene promoter and ERG results in the overexpression of N-terminally truncated or full-length forms of ERG (Klezovitch et al., 2008; Sun et al., 2008; Sreenath et al., 2011). Fusion events between ERG and other androgen inducible promoter sequences, such as SLC45A3 (Han et al., 2008) and NDRG1 have also been identified in prostate cancer (Pflueger et al., 2009; Rubin et al., 2012).
ERG expression in CaP is androgen receptor (AR) dependent. While AR signaling inhibitors are employed as therapeutics for treating CaP, compounds that selectively inhibit ERG expression are highly desirable. Up to 4 million of patients living with prostate cancer worldwide are expected to harbor ERG positive tumors (Farrell et al., 2013). Compounds such as ERGi-USU are examples of such selective inhibitors that inhibit the ERG protein in ERG positive cancer cell lines with minimal effect on normal primary endothelial cells that endogenously express ERG—i.e., ERG negative tumor or normal cells (PCT US2015/020172). The azophenols also selectively inhibit ERG expression and thus provide for the treatment of cancers or pathologic conditions associated with an ERG fusion event or ERG overexpression, including, for example, prostate cancer, Ewing's sarcoma, acute myeloid leukemia, megakaryoblastic leukemia, endothelial cancer and acute T-lymphoblastic leukemia.
A systematic screening of 456 known kinases in kinase ligand competition assays (Fabian et al., 2005) indicated potential ligands for RIO2 (Kd=200 nM). The RIO family of atypical serine/threonine kinases was first characterized in 1997 based on the studies of a right open reading frame (RIO1) gene, expressed constitutively at a low level in Saccharomyces cerevisiae (Angermayr et al., 1997). Unexpectedly, RIO kinase 2 (RIOK2) protein levels were observed to decrease in ERG expressing VCaP cells in response to the azophenols of the invention with minimal effect on RIOK2 transcript levels as assessed by whole transcriptome analyses. In the present invention, RIOK2 was investigated as a potential target of the ERG inhibitors described herein.